Smallpox virus is a major threat for bio-terrorist attacks since the majority of the population is now susceptible following its eradication from the wild. Vaccinia virus (VV) vaccine stocks are now very limited and serious concerns have been raised about the safety and long-term usefulness of VV immunization prompting urgent calls for improved vaccination strategies. Cause for further alarm is that poxviruses, among a variety of pathogens, may relatively easily be engineered to increase their virulence, often rendering host immune responses ineffective. Such 'stealth' viruses may even overcome immunity in previously vaccinated individuals. The primary focus of this application is to evaluate novel strategies against highly virulent poxviruses and against stealth viruses with a capacity to suppress host antiviral immunity. Recently we developed a consecutive DNA/poxvirus "prime-boost" protocol that induces unprecedented levels of antiviral immunity of high avidity for the immunizing antigen. T cell responses are characterized by high levels of IFNg production and are sustained for months, being rapidly activated upon re-exposure to antigen. Here, we will determine whether these special qualities render prime-boost vaccinees protection against virulent poxvirus infection. Our vaccines comprise (i) novel DNA plasmids encoding immunogenic poxvirus proteins, bearing highly adjuvant backbones, and (ii) attenuated poxviruses (VV and fowlpox). Initially, combinations of DNA/poxvirus and poxvirus/poxvirus will be tested and T and B cell responses characterized and correlated with protective efficacy after challenge with viruses of high (ectromelia, mousepox) or relatively low (VV) virulence. Next, the most immunogenic of these strategies will be tested against infection with immunosuppressive stealth viruses. To facilitate these studies, we developed a mouse model of infection with ectromelia encoding IL-4 (EV-IL-4). This virus displays remarkably increased pathogenicity through inhibition of antiviral T cell responses and, strikingly, has a high mortality rate, even in EV- and VV - immune animals. Finally, we will use this model to test whether direct neutralization of a viral virulence factor (i.e. encoded IL-4) represents an effective therapeutic strategy. This application addresses important objectives of the RFA of direct relevance to development of safer and more effective vaccination strategies against virulent poxviruses and has implications for prophylaxis of other pathogens posing grave threats as potential bio-weapons.